Washout CYFRA 21-1: A tool to improve diagnostic accuracy of fine needle aspiration in the diagnosis of metastatic lymph nodes in papillary thyroid cancer

Thyroid carcinoma has an increasing incidence of endocrine system cancers. Fine needle aspiration cytology (FNAC) and thyroglobulin (Tg) are the primary diagnostic modalities employed for assessing metastatic lymph nodes (LNs) in thyroid cancer. Due to the limited accuracy, rare patients benefited from these procedures. In this research, we aimed to discover a dependable biomarker that could increase the accuracy of FNAC's ability to diagnose metastatic LNs among patients suffering from papillary thyroid cancer (PTC). From March 2021 to July 2023, 99 LNs from PTC patients who had thyroid ultrasonography suspicions of metastases were examined. All patients underwent FNAC, washout Tg and CYFRA 21-1 measurements. Surgical histology and a subsequent FNAC were utilized to validate the outcomes of LNs. In our study, the optimal cut-off value for CYFRA 21-1 washout fluid was 1.145 ng/mL, with a specificity of 94.00 % (slightly lower than Tg and FNAC at 98 %). However, CYFRA 21-1 demonstrated significantly higher diagnostic sensitivity (85.71 %) and accuracy (86.41 %) compared to Tg (71.43 %, 81.55 %) and FNAC (69.39 %, 80.58 %). Furthermore, FNAC plus washout CYFRA 21-1 performed better in diagnosing the metastatic LNs in PTC than FNAC plus Tg, which may indicate a novel solution for metastatic LNs diagnosis in PTC.


Introduction
Papillary thyroid cancer (PTC) is the predominant pathological subtype of thyroid cancer, and 30 %-80 % of thyroid cancer patients presenting with cervical lymph node metastasis, leading to adverse patient outcomes, including tumor recurrence or mortality [1][2][3].Currently, fine needle aspiration cytology (FNAC) is extensively utilized as a diagnostic tool for PTC to identify lymph node (LN) metastasis.Nevertheless, it is operator-dependent and has a relatively low sensitivity, resulting in a certain rate of false negatives [4][5][6].Therefore, there is a pressing need to identify additional biomarkers that could be employed alongside FNAC to enhance the diagnostic accuracy in detecting LN metastasis in PTC.
Thyroglobulin (Tg), a precursor protein for thyroid hormones that are crucial for human growth and metabolic regulation, is considered to have a good diagnostic performance in LN metastasis of thyroid cancers [6][7][8].Adding Tg measurement to FNAC can increase the detection rate of PTC-associated LN metastasis by 13 % [9].However, factors such as thyroidectomy status and immunological assay methods can impact the values for Tg, resulting in current discrepancies in the cut-off values for Tg diagnosis of PTC with metastasis [10,11].The cytokeratin 19 fragment (CYFRA 21-1) is predominantly found in squamous and simple epithelial cells, and its expression is significantly increased in malignant tumors [12,13].Elevated CYFRA 21-1 levels demonstrated a significant correlation with the presence of distant metastasis in patients with thyroid cancer [14].However, the definitive role of CYFRA 21-1 in PTC still need to be explored.
The purpose of this study is to determine CYFRA 21-1's diagnostic significance, which could potentially improve the diagnostic precision of detecting metastatic LN in PTC when used in conjunction with FNAC.

Study subjects
Patients (n = 72, >18 years old) with PTC were sequentially enrolled from March 2021 to July 2023 at Shangyu People's Hospital of Shaoxing.The diagnosis of PTC was established according to published criteria [2].All enrolled patients meet the following conditions: 1) Postoperative histopathological confirmation of primary lesion as PTC, 2) FNAC was performed before surgery, 3) Complete clinical data, including preoperative and postoperative follow-up ultrasound examination, FNAC information, surgical pathology, preoperative washout Tg, and wash out CYFRA 21-1.Exclusion criteria: 1) Postoperative cytologic result is non-PTC; 2) The patient has not undergone neck dissection.

Washout Tg and CYFRA 21-1 measurements
99 LNs from 72 patients were examined in this study.Among these patients, 50 had a single lymph node punctured, 17 had two lymph nodes punctured, and 5 had three lymph nodes punctured.All LNs underwent fine needle aspiration, subsequently undergoing analysis of the aspirated washout for the detection of Tg and CYFRA 21-1 by automatic electrochemical luminescence analyzer (Roche Diagnostics) [15].

US and FNAC of suspicious LNs
The suspicious LNs underwent ultrasound (US)-guided FNAC through an EPIQ 7 sonography system (Philips Medical Systems, Bothell, USA), which was administered by experienced radiologists.LNs were considered suspicious if they exhibited characteristics described in the previous study [16].Following aspiration, samples were prepared for hematoxylin and eosin (HE) stains as well as Papanicolaou stains.

FNAC and LN assessment
The FNAC was operated by experienced pathologists, and LNs were categorized into two groups according to the postoperative histopathological results: metastatic LN group and benign LN group.Metastatic LN group: FNAC or histopathology were positive.Benign LN group: both histopathology and FNAC were negative.
In this study, the diagnosis of surgical pathology results is considered the golden standard.The sample size calculation formula suggests a required sample size of 48.98 for the metastatic LN group and 34.57for the benign LN group [17].

Statistical analysis
Student's t-test was used to determine statistical significance.The optimal cut-off values were confirmed through the receiver operating characteristic (ROC) curve analysis.P values of 0.05 were considered statistically significant in all analyses conducted with SPSS 22.0 software.

Overview of study subjects
Among 72 patients, 36 patients exhibited no LN metastasis, while the remaining 36 patients had LN metastasis.A total of 8 thyroid tumors smaller than 1 cm were found in the 36 patients with LN metastasis.As indicated in Table 1, regarding age, gender, TSH, and hypertension status, no discernible differences were observed between the two groups.Importantly, the level of Tg was increased in the metastatic group (Fig. 1A).In the metastatic group, CYFRA 21-1 expression was also elevated (Fig. 1B).

Diagnostic performance of FNAC, Tg and CYFRA 21-1
According to surgical pathology results, the diagnostic performance has been summarized in Table 2.Among 49 metastatic LNs, FNAC missed 15, whereas washout Tg and washout CYFRA 21-1 failed to detect 14 and 7, respectively.Within the 50 benign LNs, both FNAC and washout Tg erroneously classified 1 LN as metastatic, whereas washout CYFRA 21-1 misclassified 3 LNs.
C. Huang et al. accuracy (86.41 %) has not been significantly improved.

Discussion
Timely diagnosis and standardized treatment can achieve a high disease-free survival (DFS) rate for the majority of PTC patients [18].Nowadays, ultrasound-guided FNAC has gained widespread acceptance in thyroid surgery [19].However, the accuracy of FNAC was not ideal in some thyroid tumors, and histology was often needed for diagnosis [5].Additionally, the limitations of FNA detection also manifested in the diagnosis of larger thyroid nodules (diameter >3 cm) [20].Thus, if combined with molecular diagnostic methods, the detection range of FNA in thyroid nodules can be improved.
In thyroid cancer, FNA Tg assessment was introduced in 1992 and has demonstrated its superiority in sensitivity compared to FNAC.Its integration with FNAC enhances diagnostic precision and sensitivity [21,22].Although diagnostic sensitivity is increased, the diagnostic threshold remains undetermined, and interference from elevated serum anti-thyroglobulin antibodies (TgAb) levels may lead to false-negative outcomes [10].Cytokeratin 19 (CK19) is part of the epithelial cytoskeleton, with tissue overexpression in differentiated thyroid carcinoma (DTC), particularly in cases of papillary carcinoma [23].Soluble CK19 fragments, known as CYFRA 21-1, released by cancer cells can serve as diagnostic or prognostic markers for lung, intrahepatic cholangiocarcinoma, and breast cancers [24][25][26].According to a previous study, thyroid tumors with distant metastases may have higher CYFRA 21-1 levels than thyroid malignancies without metastases [14].In patients with DTC, CYFRA 21-1 can serve as an indicator for prognostic assessment [27].In our study, metastatic LN patients had elevated levels of washout Tg and CYFRA 21-1, aligning with previous findings.Furthermore, combining FNAC and CYFRA 21-1 exhibited improved diagnostic performance compared to FNAC alone in detecting metastatic pleural fluids from adenocarcinomas [28].Moreover, our study found that washout fluid with CYFRA 21-1 at 1.145 ng/mL had the best cutoff value, exhibiting a specificity of 94.00 %, slightly lower than the 98 % specificity of Tg.However, its diagnostic sensitivity was 85.71 %, significantly surpassing the 71.43 % sensitivity of Tg.Besides, combining CYFRA 21-1 with FNAC showed enhanced sensitivity, diagnostic accuracy and NPV compared to the other approaches, such as FNAC, Tg, and FNAC plus Tg assessment.Notably, the combination of CYFRA 21-1, FNAC, and Tg achieved the highest sensitivity (89.80 %) and NPV of 90.00 % compared to other evaluated methods, indicating that CYFRA 21-1 could potentially improve FNAC's diagnostic performance for LN metastasis prediction.
There are some limitations to our study, such as its relatively small sample size, which necessitates further investigation on a larger cohort to validate our findings.Additionally, we lack long-term follow-up data on patients which could offer insights into the accuracy of molecular diagnostics and the efficacy of treatments.

Table 1
Clinical characteristics of 99 lymph nodes were evaluated in the study.

Table 2
Individual diagnostic performance of FNAC, Washout Tg, and Washout CYFRA 21-1, and their combination according to the outcomes.

Table 3
Comparison of diagnostic values of FNAC, washout Tg, washout CYFRA 21-1 and their combinations.